Human toxicities from dietary exposure to toxic metals such as cadmium (Cd) contribute to risks of many chronic and age-related diseases in the U.S. population. Phytochelatins (PyCs) are metal-binding plant compounds commonly consumed in the human diet which may protect from absorption of toxic metals in the diet. However, little is known about the concentrations of PyCs in plant foods and their impact on metal uptake and cellular responses in the intestine. The purpose of this proposal is to characterize PyC types and concentrations in human foods and determine the impact of PyCs at relevant dietary concentrations on Cd uptake and transport in intestinal cells. We focus on Cd as average dietary Cd intake is linked to disease in multiple organ systems and is known to impact intestinal cell proliferation and differentiation, inflammatory signaling, and oxidative stress. Additionally, Cd increases risks of gastrointestinal diseases such as colitis. The project is based on characterization of PyCs in the plant biology literature, our preliminary data demonstrating PyCs can be measured in commonly consumed plant foods, and extensive literature on the biological effects of Cd. In initial research, we developed a PyC database of 46,000 PyC and PyC-metal complexes and a web-based tool to facilitate research of PyCs and PyC-metal complexes. In commonly consumed plant foods, we will determine the types and relative concentrations of PyCs (Aim 1) using mass spectrometry-based metabolomics and the PyC database. To understand the role of PyCs at relevant dietary concentrations, we will first investigate the impact of varying concentrations of one of the most common PyCs in plants, PyC2-Gly, on uptake and transport of PyCs and Cd in the human intestinal epithelial cell line, Caco-2 (Aim 2). Using the cellular metabolomics data we generate, we will determine how PyC and Cd absorption impacts changes to metabolites and metabolic pathways in intestinal cells (Aim 2). The results will provide the first dose-response characterization of PyC effects on Cd transport and intestinal cell metabolism. Through completing these complementary Aims, we will significantly advance our understanding of the major dietary contributors to PyC exposure and the role of PyCs in Cd and PyC uptake and transport in the intestine. The long-term goal of this project is to determine whether PyCs could be responsible for large differences in absorption of toxic metals in the diet. Understanding how PyCs impact toxic metal uptake and transport will help lead to improved dietary recommendations to mediate toxic metal exposure.